Method for treating the central nervous system by administration of igf structural analogs

ABSTRACT

This invention is directed to a method for treating a disease or disorder of the brain or spinal cord in a mammal, including human, comprising the administration of an effective amount of an insulin-like growth factor (IGF) structural analog at a site outside of the blood-brain, blood-central nervous system, or blood-spinal cord barrier.

This patent application is based on provisional U.S. patent applicationSer. No. 60/228,633, filed Aug. 29, 2000.

The United States Government may own certain rights in the presentinvention pursuant to National Institute of Neurological Disorders andStroke Grants Nos. 5 RO1 NS24327 and 9 RO1 DK53922, as well as Centersfor Disease Control Grant R49 CCR811509.

SUMMARY OF THE INVENTION

This invention is directed to a method for treating the central nervoussystem by the nonintracranial and nonintravertebral columnadministration of one or more IGF structural analogs. More particularly,the invention is directed to a method for treating disorders or diseasesof the brain or spinal cord by the nonintracranial or nonintravertebralcolumn administration of one or more IGF structural analogs.

DESCRIPTION OF DRAWINGS

FIG. 1. Concentration-dependent detection of (A) hIGF-I, (B)Des(1-3)hIGF-I, (C) [Leu²⁴]hIGF-I and (D) [Leu⁶⁰]hIGF-I by ELISA.Samples were assayed in triplicate at each concentration. Thecoefficient of correlation, r, was determined by linear regression usinga computer software program.

FIG. 2. Dose-dependent distribution of immunoreactive hIGF-I in CSF andplasma following subcutaneous injections in adult rats. Plasma and CSFwere withdrawn for ELISA 90 min after a single bolus subcutaneousinjection of the indicated dose of hIGF-I, and each sample was assayedin triplicate. Group means±SEM are shown (n=3 rats per dose). Part A,CSF hIGF-I. Part B, plasma hIGF-I. The data were plotted using linearregression, r=0.97.

FIG. 3. Effect of simultaneous administration of hIGF-II on hIGF-Iuptake into CSF. Rats were injected subcutaneously with 150 μg hIGF-Ialone (n=8) or the combination of 150 μg hIGF-I and 400 μg hIGF-II(n=6). Plasma and CSF were withdrawn 90 min later for assay. Values aremeans±SEM. The group means were compared using a t-test. *P<0.02.

FIG. 4. Comparative distribution in CSF and plasma followingadministration of Des(1-3)hIGF-I (n=4), hIGF-I (n=3), or vehicle (n=2).Equivalent amounts (200 μg per rat) of Des(1-3)hIGF-I or hIGF-I wereinjected subcutaneously, and plasma and CSF were withdrawn for assay 90min later. Group means were compared using Newman-Keuhl's posthoc test.*P<0.002 and 0.003 for Des and hIGF-I, respectively, vs. control in CSF.*P<0.002 for hIGF-I vs. control in plasma.

FIG. 5. Uptake of [Leu²⁴]hIGF-I and [Leu⁶⁰]hIGF-I into CSF.[Leu²⁴]hIGF-I (200 μg per rat; n=3 rats), [Leu⁶⁰]hIGF-I (100 μg per rat;n=4) or hIGF-I (200 μg per rat; n=3) or vehicle (n=9) were injectedsubcutaneously, and 90 min later plasma and CSF were withdrawn forassay. Part A, CSF; Part B, plasma. Differences between group means weredetected using Newman-Keuhl's posthoc test. *P<0.002 for hIGF-I vs.[Leu²⁴]hIGF-I in CSF. *P<0.0004 for [Leu²⁴] and [Leu⁶⁰] vs. control and0.0007 for hIGF-I vs. control in CSF. In plasma, *P<0.0002 and 0.0005for [Leu²⁴] and [Leu⁶⁰], respectively, vs. hIGF-I. *P<0.0005 and 0.0002for [Leu⁶⁰] and hIGF-I, respectively, vs. control.

DETAILED DESCRIPTION OF THE INVENTION

This invention is directed to a method for treating the central nervoussystem by the nonintracranial and nonintravertebral columnadministration of one or more IGF structural analogs. More particularly,the invention is directed to a method for treating disorders or diseasesof the brain or spinal cord by the nonintracranial or nonintravertebralcolumn administration of one or more IGF structural analogs. Forpurposes of this invention, “IGF structural analogs” are defined asmolecules having substantial sequence homology to naturally occurringinsulin-like growth factors (IGFs), including human and animal(including but not limited to cow, pig, dog, sheep, horse, deer, goat,rat, mouse and chicken) IGF-I and IGF-II. More preferably, the IGFstructural analogs have amino acid sequences of IGF molecules that havebeen modified by deletions, substitutions and/or additions of fewer than15 amino acids.

In the method according to the invention, the preferred route ofadministration of the IGF structural analog is from a site outside ofthe blood-brain-barrier (BBB), blood-central nervous system-barrier(B-CNS-B) and blood-spinal cord-barrier (B-SC-B). Any of the commonroutes of administration known to the pharmaceutical sciences may beused that can deliver IGF structural analogs into the circulation,including but not limited to percutaneous, intradermal, subcutaneous,intravenous, intramuscular, intraarterial, intraperitoneal, parenteral,buccal, sublingual, rectal, oral, nasal, by inhalation, from asubcutaneous implanted pump or matrix, or from a plasmid constructcontaining an IGF structural analog gene that is administered at a siteoutside of the BBB, B-CNS-B and B-SC-B. For example, the nasal cavityand lung are richly vascularized, and IGF structural analogsadministered into the nasal cavity or by inhalation may be rapidly takenup by the local microvasculature, resulting in IGF analogs being takenup into cerebrospinal fluid (CSF) across the BBB or B-CNS-B. Thisinvention is not limited to a particular route of administration, otherthan that the administration is from a site outside of the BBB, B-CNS-Band B-SC-B.

In a preferred embodiment, an IGF structural analog may be administeredalone or in combination with other IGF analogs. The IGF analog may alsobe combined with one or more excipients, coloring agents, salts,solvents, carriers, stabilizers, and other ingredients that may be usedin formulations and are known to the pharmaceutical sciences. In afurther preferred embodiment, the IGF structural analog is administeredin an amount from about 0.01 μg/kg/day up to about 4 mg/kg/day.

In a preferred embodiment, the invention is directed to a method fortreating disorders or diseases of the postbirth brain or spinal cord,such as Alzheimer's Disease, Parkinson's Disease, AIDS-related dementia,senile dementia, stroke, trauma, cortical-basal ganglionic syndromes,progressive dementia, familial dementia with spastic paraparesis,progressive supranuclear palsy, multiple sclerosis, hepaticencephalopathy, Pick's Disease, Huntington's Disease, diffuse cerebralsclerosis of Schilder, acute necrotizing hemorrhagic encephalomyelitis,brain tumors and the like. This invention does not include amyotrophiclateral sclerosis.

IGF structural analogs that may be used in the present invention includebut are not limited to des(1-3)IGF-I, which is an IGF-I analog lackingthe N-terminal tripeptide; [Arg3]IGF-I, which is an IGF-I analog inwhich Arg is substituted for Glu at position 3; [Leu24]IGF-I, which isan IGF-I analog in which Leu is substituted for Thr at position 24;[Leu60]IGF-I, which is a mutant IGF-I with Leu substituted for Tyr atposition 60; Long R3IGF-I, which is a mutant IGF-I with Arg substitutedfor Glu at position 3 as well as a 13 amino acid extension at theN-terminus; des(1-6)IGF-II, which is an IGF-11 analog lacking theN-terminal hexapeptide; [Gly1]IGF-II, which is an IGF-11 mutant with Glysubstituted for Ala at position 1; [Arg6]IGF-II, which is an IGF-IImutant with an Arg substituted for Glu at position 6; and [Leu27]IGF-II,which is an IGF-IL mutant with Leu substituted for Tyr at position 27.These IGF structural analogs are commercially available, for example,from GroPep, Pty. Ltd. (Australia). It is appreciated that in the art itis possible to produce various additional IGF structural analogs.

The IGF structural analogs used in the present invention have biologicalactivity. For example, it is known that des(1-3)IGF-I administered intothe eye can enhance the growth of transplanted spinal cord, cerebralcortex and parietal cortex in the eye. It can increase cholineacetyltransferase activity in cultured spinal cord and enhance growth ofcultured olfactory bulb cells. [Arg3]IGF-I, long R³IGF-I, [Leu24]IGF-I,[Leu60]IGF-I, des(1-6)IGF-II, [Gly1]IGF-II, [Arg6]IGF-II, and[Leu27]IGF-II can bind to type I IGF receptors, type II IGF receptors,or IGF binding proteins and alter protein synthesis in cells. Thus, IGFstructural analogs that cross the BBB, B-CNS-B or B-SC-B may be used forthe purposes of this invention.

The treatment of the brain and spinal cord is more complicated than thetreatment of the peripheral nervous system because the B-CNS-B, B-SC-Band BBB pose an obstacle to the delivery of pharmaceutical agents,particularly proteins and peptides, to the central nervous system. Thesebarriers are widely believed to prevent the uptake and penetration ofproteins and peptides, such as IGFs, and these concerns would applyequally well to IGF structural analogs. Applicant has previously shownthat IGF-I or IGF-II can cross from the blood into the CSF and normalizebrain biochemistry in disease, prevent loss of axons in the spinal cord,and prevent functional damage to the central nervous system. Therefore,based on subsequent research, it is expected that IGF structural analogscan likewise cross from the blood into the cerebral spinal fluid (CSF)and may prevent damage, disease or disorder in the central nervoussystem.

The following examples show that IGF structural analogs can enter theCSF from the circulation. Consequently, IGF structural analogs mayeffect changes in or treat the central nervous system. The examples showthat there is a carrier that takes IGFs up from the circulation intoCSF, and the properties of this carrier differ from known IGF bindingproteins and IGF receptors, such as type I IGF receptor or type II IGFreceptor. The IGF analogs in the examples that are taken up into CSFincludes des(1-3)IGF-I, [Leu24]IGF-I and [Leu60]IGF-I. Furthermore,IGF-II reduces IGF-I uptake into CSF, and this is consistent withcompetition for uptake by a common IGF carrier. The invention in itsbroader aspects is not limited to the specific details or representativeexamples described. Therefore, based on subsequent research, it isexpected that IGF structural analogs that are taken up into CSF by thiscarrier may be used for the purposes of this invention. Those IGFstructural analogs that are taken up into CSF may serve as agonists orantagonists. Antagonists may be useful for inhibiting the growth ofbrain tumors that may be IGF-dependent, for example. Agonists may beuseful for treating various brain diseases and disorders such asParkinson's Disease, Alzheimer's Disease, multiple sclerosis, stroke,trauma, senile dementia, and the like.

In the examples, IGF structural analogs were injected subcutaneouslyinto rats. Ninety minutes later plasma and CSF were withdrawn andanalyzed by an ELISA (Table I). TABLE 1 Selective detection of hIGF-Iand its analogs by ELISA OD (450) Sample Mean ± SEM P Value Blank  0.0 ±0 Human IGF-I (150 pg) 0.568 ± .113 0.001 Des (1-3) hIGF-I (150 pg)0.276 ± .047 0.001 Leu 24 hIGF-I (150 pg) 0.661 ± .072 0.001 HumanIGF-II (150 pg) 0.004 ± .017 0.959 Insulin (150 pg) 0.016 ± .006 0.903Rat CSF (extracted) 0.018 ± .034 0.971 Rat Plasma (extracted) 0.051 ±.037 0.818hIGF-I and other proteins were subjected to the ELISA shown in FIG. 1.Untreated rat CSF and plasma were tested at the same volumes assayedthroughout these experiments. Note that rat IGF-I, IGF-II, insulin andIGFBP in CSF and plasma do not interfere in the ELISA. Values are means± SEM of four replicate measurements.

The ELISA detected human IGF-I and IGF structural analogs. However, theELISA did not detect IGF-II or insulin. Furthermore, nothing inuntreated rat CSF or plasma interfered with the ELISA, showing that thistest was specific for human IGF-I and IGF structural analogs. In otherwords, endogenous rat IGF-I, rat IGF-II, rat insulin and other ratsubstances in CSF or plasma did not interfere in the ELISA. FIG. 1 showsstandard ELISA curves for different concentrations of human IGF-I,des(1-3)IGF-I, [Leu24]IGF-I and [Leu60]IGF-I.

Adult rats were injected subcutaneously with various doses of humanIGF-I. FIG. 2 shows that IGF-I in plasma increased linearly with dose.However, IGF-I uptake into CSF saturated with increasing dose, showingthat uptake was via an IGF uptake carrier. FIG. 3 shows that IGF-IIcompeted with IGF-I for uptake into CSF.

EXAMPLES Example 1

Des(1-3)IGF-I is missing the first 3 amino acids from the N-terminusresulting in at least 25-fold reduced affinity for IGF binding protein-3(IGFBP-3), IGFBP-4 and IGFBP-5. Binding to IGFBP-1 is reduced as well.Des(1-3)IGF-I binds to the type I IGF receptor, and has enhancedbiological activity on neurons. It is more potent due to reduced bindingto IGFBP. FIG. 4 shows that des(1-3)IGF-I administered subcutaneously istaken up into cerebrospinal fluid in adult rats. Therefore, binding ofIGF and mutant IGFs to IGFBP-1, -3, 4 and -5 is not required for uptakeinto CSF, and the IGF uptake carrier molecule does not havecharacteristics of IGFBP-1, -3, -4 or -5.

Example 2

Leu is substituted for Thr at position 24 in [Leu24]IGF-I. Followingsubcutaneous injection of [Leu24]IGF-I into adult rats, it was readilydetected in cerebrospinal fluid (FIG. 5). This, together with Examples 1and 3, shows that IGF structural analogs with various deletions orsubstitutions can be taken up into CSF from the circulation.

Example 3

Leu has been substituted for Tyr at position 60 in [Leu60]IGF-I, whichhas a 20-fold reduced affinity for the type I IGF receptor. Followingsubcutaneous injection of [Leu60]IGF-I into adult rats, it was readilydetected in cerebrospinal fluid (FIG. 5). This shows that binding to thetype I IGF receptor is not necessary for uptake of IGFs, and the IGFcarrier molecule does not have characteristics of the type I IGFreceptor.

Des(1-3)IGF-I and IGF-I do not bind appreciably to the type II IGFreceptor, yet both of these ligands are taken up into CSF followingsubcutaneous administration. Thus, binding to the type II IGF receptoris not required for uptake of IGFs into CSF, and the IGF carriermolecule does not have characteristics of the type II IGF receptor.

Uptake of insulin-like growth factors (IGFs) from the circulation intocerebrospinal fluid (CSF) is consistent with a transport carrierprotein. This carrier protein does not have the same properties as thetype I or type II IGF receptors, or IGF binding proteins. Consequently,the carrier has properties unlike that of previously characterized IGFbinding molecules.

Therefore, IGF structural analogs are shown to enter the CSF from acrossthe BBB, B-CSF-B and/or B-SC-B in a mammal. This invention has theadvantage that mutant IGFs and IGF analogs may be administered fromoutside of the BBB, B-CSF-B and B-SC-B, and it would not be necessary touse invasive and riskier methods of administration such as intracranialor intrathecal. The risk and cost of surgery and risk of CNS infectionmay be circumvented by the invention.

1. A method of treating the central nervous system, comprisingadministering an effective amount of an IGF structural analog to treator prevent neuronal damage in the central nervous system.
 2. The methodof claim 1, wherein the neuronal damage to the central nervous system isdue to a disorder or disease in the post-birth brain or spinal cord. 3.The method of claim 2, wherein the neuronal damage in the brain is dueto Alzheimer's Disease, Parkinson's Disease, AIDS-related dementia,senile dementia, stroke, trauma, cortical-basal ganglionic syndromes,progressive dementia, familial dementia with spastic paraparesis,progressive supranuclear palsy, multiple sclerosis, hepaticencephalopathy, Pick's Disease, Huntington's Disease, diffuse cerebralsclerosis of Schilder, or acute necrotizing hemorrhagicencephalomyelitis.
 4. The method of claim 2, wherein the neuronal damagein the brain or spinal cord is a tumor or cancer.
 5. The method of claim1, wherein the IGF structural analog is des(1-3)IGF-I.
 6. The method ofclaim 1, wherein the IGF structural analog is [Arg3]IGF-I, [Leu24]IGF-I,[Leu60]IGF-I, Long R3IGF-I, des(1-6)IGF-II, [Gly1]IGF-II, [Arg6]IGF-IIor [Leu27]IGF-II.
 7. The method of claim 1, wherein the mutant IGF orIGF analog is administered in an amount from about 0.01 μg/kg/day up toabout 4 mg/kg/day.
 8. The method of claim 1, wherein the IGF structuralanalog is administered by nonintracranial and nonintravertebral columnadministration.
 9. A method of treating the central nervous system,comprising nonintracranial and nonintravertebral column administrationof an effective amount of an IGF structural analog to treat or preventneuronal damage in the central nervous system.
 10. The method of claim9, in which the damage is due to a disorder or disease in the centralnervous system, except where the disease is amyotrophic lateralsclerosis.
 11. The method of claim 9, wherein the damage is due to atumor or cancer.
 12. The method of claims 9, wherein the mutant IGF orIGF analog is des(1-3)IGF-I.
 13. The method of claim 9, wherein the IGFstructural analog is [Arg3]IGF-I, [Leu24]IGF-I, [Leu60]IGF-I, LongR3IGF-I, des(1-6)IGF-II, [Gly1]IGF-II, [Arg6]IGF-II or [Leu27]IGF-II.14. The method of claim 9, wherein the mutant IGF or IGF analog isadministered in an amount from about 0.01 μg/kg/day up to about 4mg/kg/day.
 15. The method of claim 9, wherein the nonintracranial andnonintravertebral column administration is percutaneous, subcutaneous,intramuscular, intravenous, intraarterial, by inhalation, or intranasal.